The long term objective of this research is to elucidate the extrahelical base recognition mechanism used by DNA repair glycosylases to detect and excise damaged bases in genomic DMA. This work relates to the DNA repair processes involved in preserving the integrity of the genome, which impacts human health in many ways, including the prevention of cancer and inheritable genetic diseases. We will use novel NMR and crystallographic methods to uncover the important structural and dynamic aspects of this recognition mechanism termed "base flipping". A key goal is to determine whether enzymes passively capture, and then interrogate, normal and damaged bases that emerge from the DNA duplex because of thermally induced base pair breathing motions, or alternatively, whether enzymes use active mechanisms to promote damaged base expulsion from the DNA stack. The difference is critical. For the former, the structure and dynamics of the damaged site initiates its own repair, and for the latter, the enzyme provides the essential means for ejecting the base. The specific aims are (i) to develop a new chemical approach called "reaction coordinate tuning" to trap an otherwise energetically unstable extrahelical intermediate that occurs very early on the pathway for thymine and uracil flipping by uracil DNA glycosylase. This structure will uncover the earliest interactions that the enzyme uses to promote base flipping, (ii) Directed by the structure, site- directed mutagenesis will be used to delete enzyme side chains involved in stabilizing the extrahelical conformation. The effects of the mutations on the dynamics of the flipped base will be measured using our recently developed NMR imino proton exchange methods. These DNA dynamic measurements will elucidate the detailed mechanism of base pair opening. (Hi) Use NMR relaxation methods to measure the dynamics of enzyme backbone NH groups involved in the earliest step in extrahelical recognition. The enzyme motions must be rapid enough to efficiently trap the uracil in the life-time of its extrahelical state. Hence, DNA dynamics will be linked to enzyme dynamics. Public Health Relevance: DNA bases are damaged hundreds of time each day in every cell of the human body. Without extraordinary enzyme machines to locate and repair these damaged sites, cancer and disease would be rampant. The goal of this work is to elucidate how these machines locate these sites, with the long term goal of using the recognition principles to design molecules that efficiently direct these enzymes to specific damaged sites in the genome.